Novel diagnostic system for differentiation of enterobacteriaceae

ABSTRACT

This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.

United States Patent [1 1 Warren et al.

[ 1 NOVEL DIAGNOSTIC SYSTEM FOR DIFFERENTIATION OF ENTEROBACTERIACEAE[75] Inventors: Bert Warren, Tuxedo Park, N.Y.; George L. Evans,Hopatcong, NJ.

[73} Assignee: Schering Corporation, Bloomfield,

[22] Filed: May 4, 1973 [21] Appl. No.: 357,318

[52] US. Cl. 195/1035 R, 195/100 [51] Int. Cl Cl2k 1/04 {58] Field ofSearch 195/99-103.5 R

[' r 3,870,601 Mar. '11, 1975 Primary Examiner-Samih N. ZaharnaAssistantExaminer-Robert J. Warden Attorney, Agent. or Firm-Raymond A.McDonald; Stephen B. Coan [57] ABSTRACT This invention relates to aculture medium for the rapid differentiation and identification ofbacteria belonging to the family Enterobacteriaceae.

18 Claims, No Drawings NOVEL DIAGNOSTIC SYSTEM FOR DIFFERENTIATION OFENTEROBACTERIACEAE This invention relates to a culture medium for therapid differentiation and identification of bacteria of the familyEnterobacteriaceae. More specifically, this invention relates to singleculture media, the method for their preparation and the utilization ofthese media for the identification of the bacteria of the familyEnterobacteriaceae.

The Enterobacteriaceae are a ubiquitous group of bacteria consisting offrank enteric pathogens (Salmonella and Shigella) and many otheropportunistic organisms capable of causing infections in everyconceivable body locus. They are defined as gram negative rods thatreduce nitrates, are oxidase negative and ferment glucose. Theirspectrum of sensitivity to antibiotics varies considerably and in manyinstances therapy ought be based only upon the identity of the organism.Some members are epidemiologically significant and all requireidentification for specific diagnosis. Currently, they are the mostfrequent cause of bacterial infections and account for over milliontests per year.

Although certain individual principles and aspects to which thisinvention relates have been known and used in hospital and fieldconditions for the determination of bacteria, such factors havegenerally employed the use of multiple media requiring at least threedays and as many as 10-20 separate tests. Although other procedures havebeen produced to obviate some of the problems involved, such procedureshave been complex, awkward and expensive multi-media devices. The mediaof this invention provide a relatively simply prepared single confluentmedium which affords the diagnostician with a large number ofdifferential tests in a single tube. Other advantages and distinguishingcharacteristics of this invention over the media of the prior art willalso be apparent to the skilled artisan as the details of this inventionare explored.

1n its broad concept this invention relates to media wherein chromogenicB-galactoside substrates are admixed with either (a) a decarboxylasesubstrate, (b) a deaminase substrate, (c) a urease substrate, (d) ahydrogen sulfide detecting system, or (e) a carbohydrate fermentationsystem, or the chromogenic B-galactoside substrate is admixed with anycombination of such systern.

Suitable chromogenic B-galactoside substrates areo-nitrophenyl-,B-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-,B-galactoside, and 6-bromo-2-naphthyl-B-D-galactoside, as well as any other wellknown agents.Suitable deaminase substrates are such l-amino acids as phenylalanine,tryptophane, histidine, leucine, norleucine, methionine and norvalineand the like. Urea is used as the substrate for urease. Suitablehydrogen sulfide detecting agents are sodium thiosulfate in the presenceof an iron containing salt such as ferric ammonium citrate. Suitabledecarboxylase agents are lysine, ornithine and arginine, and the like.Suitable fermentable carbohydrates (or sugar alcohols) are dextrose,mannitol, arabinose, sucrose, dulcitol, rhamnose, and the like. Thesurprising feature of this invention, as the below described media andcolor formations indicate, is that for the first time a system has beendevised wherein a chromogenic B-galactoside substrate has been combinedwith the abovementioned other type substrates without the colorreactions of the chromogenic ,B-galactoside substrate interfering-withthe efficacy of the tests. Also. as is evident from the herein describedmedia, although it is preferred to strive for as many substrates aspossible. it is of course understood that such media can be modifiedwherein one or more of the above-described a, b, c, d. or e componentscan be eliminated from the medium.

Essentially the preferred medium of this invention is comprised of suchingredients as bromthymol blue (used as pH indicator), yeast extract (asource of nutrient), dextrose and/or other fermentable carbohydrates,l-lysine (detection of lysine decarboxylase), ferric ammonium citrateand sodium thiosulfate (detection of hydrogen sulfide production),tryptophan, (detection of deaminase and indole), o-nitrophenyl,B-galactopyranoside (ONPG) (detection of B-galactosidase activity),trace amounts of lactose to activate the B-galactosidase system, ureafor detection of urease and agar as a supporting base, and sodiumchloride (for osmotic control). Optionally, starch or carboxymethylcellulose (or any other cellulose) may be added to enhance gas formationand to prolong the shelf life of the medium.

More specifically, the preferred medium is comprised and prepared asfollows:

After weighing the components of part A of this medium, sufficientdistilled water is added to bring the volume, to 700 ml. The resultingsuspension is then heated and with the aid of a magnetic stirrer isbrought into solution. The pH of the solution is adjusted to about 6.9,after which it is autoclaved at 15 psi. for 15 minutes. The remainingcomponents of the medium, (B) are brought to about pH 6.9 and sterilizedby filtering through a Nalgene filter. Both (A) and (B) components arethen admixed under sterild conditions. The final pH is adjusted to about7.0. The medium is then dispensed in suitable quantities in sterilescrew-capped tubes, a1- lowing the agar to cool while the tubes areangled to obtain a butt and slant configuration according to standardtechniques. The inside of the screw-cap is fitted with ap-dimethylamino-benzaldehyde-impregnated paper disc previously preparedaccording to standard techniques suitable for the detection of indole.

Although the foregoing formulation is the most preferredingredient-concentration, quite naturally modifications may be made toachieve substantial, but varying degrees of success. Thus, it iscomtemplated that the foregoing ingredlent-concentrations may bemodified and still be within the spirit of this invention, as follows:Bromthymol blue (0.025-O.15 g/l) yeast extract 1 .59 g/l) dextrose(0.5-5 g/l) l-lysine (5.0-20.0 g/l) ferric ammonium citrate (0. ll .2g/l) sodium thiosulfate (0.1-1.2 g/l) tryptophan (1.2l0.5 g/l), agar(10-20 gms) urea (0.5-1.5 g/l) with the adjustment to the pH to about6.7-7.1.

The use of the foregoing media presumes the primary isolation ofcultures of pure cultures. When used, the butt is stabbed and the slantstreaked with the test cul ture in the usual manner that is used toinoculate other tubed media having a slant and butt, and the screw caploosely replaced onto the tube. Tne inoculated culture is permited togrow at 37C for about 24 hours and read within 24-72 hours frominoculation. Of course, it is possible to permit the inoculated cultureat temperatures below 37C but in such instances the growth period isproportionately lgnger.

The media provide for possible color differentiation wherein theproteus-providence group of organisms give rise to a brown slant,hydrogen sulfide-producing organisms causing a blackening in the butt,lysine positive organisms giving a green butt, lysine negative organismsgiving a yellow butt, urease positive organisms give a blue-green toblue color in the butt or at the butt/slant juction. In the case ofProteus species, this bluish color will mask the lysine reaction whichis not essential for the identification of this group in the presence ofurea. The ONPG reacting organisms give rise to a green color in theslant while ONPG negative organisms turn the slant blue. Thepdimethylaminobenzylaldehyde impregnated paper disc turns red to showthe presence of indole.

The expected reactions of the above-exemplified medium are as follows:

Glucose: lf lysine is not decarboxylated and glucose is fermented, thepH of the butt will drop below 6.2, resulting in yellow color. However,if the organism is strongly urease positive, an overriding alkalinereaction ONPG: lf the organism has an inducible ,B-galactosb dase,galactose is split from ONPG liberating the yellow-colloredo-nitrophenol. lo the presence of the blue slant that forms in thismedium if no deaminase activity is present, the combination of theyellow o-nitrophenol and blue, produce a green colored slant. Blueslants occur if the organism tested is both ONPG and deaminase negative.

Urea: If the organism produces urease. ammonia is formed causing a risein pH above 7.0. With strong urease producers such as Proteus species,the color of the butt will turn blue/green to blue. Due to the deaminaseactivity combined with urease activity with Proleus' species, the slantwill turn blue/green or green/brown (olive). With weaker ureaseproducers such as Klebsiella, a blue/green color may only be produced atthe buttslant junction.

lndole: lf indole.is formed as a result of tryptopha nase activity, thedisc insert in the cap will turn red to violet. It will remain colorlessif indole is not produced.

The following tables illustrate the identification of the organisms ofthe Enterobacteriaciae.

Table I Group I: Hydrogen Sulfide Positive H 5 Tryptophan lndole LysineONPG UREA Arizona Eclwardriellu Salmonella P. mirabilis P. vulgarisCilrobacler freululir' d Group ll: Tryptophan Positive (excluding GroupI organisms) H 5 Tryptophan lndole Lysine ONPG UREA l. nmrganii I.rt'llgz'rl 1 Provide/win Group lll: lndole Positive (excluding Groups land ll organisms) H- S Tryptophan lndole Lysine ONPG URliA E. (all dSlrigella or Klebriellu or d Group IV: lndole Negative S Tryptophanlndole Lysine ONPG Urea Shigella or Salmonella I Cirrobacler dKlebsiella or d E. (loacue d E. acrogvnes 1:. liufniae or Bl E.liqm'fut'it'nx or or d Scrramu d Different biochemical types. -Withexception of Shigellu .rumrci or majority positive.

or majority negative.

COLOR REACTIONS OF ENTEROBACTERIACEAE WITH EXAMPLE I MEDIUM OrganismsSlant Butt lndole l) Irm'idenciu Brown Yellow (2) I. re/lgeriBrownlBlue-Olive Blue (3) P. morganii Brown/Blue-Olive Blue (4) l.miruhilis Brown/Blue-Olive Blue/Black (5) I. rulgurii Brown/Blue-OliveBlue/Black l (l l K lvhiivllu Green Blue/Yellow (7 ('umlmilvr jmlm/iiGreen Black/Yellow (X Ari mm Green Black/Green (*1) Iii/wurrlsir'I/uBlue Black/Green I0) I-.'. mli Green Green or Yellow (H1 Si'rru/iu GreenGreen l2) l2. liqmfucicus Blue or Green or Green Yellow l3) l5.m'rqgcnes Green Green (14) If. (/Oflflll Green Yellow 15) S. smim'iGreen ellow (no gas) (I6) Salmonella Blue Black l7) Sliigellu BlueYellow or (18) If. lltlfllllll. Blue or Green Green Two color in thebutt.

Numbers cross-correlate with FIGS. l and 2.

We claim:

1. A culture medium having a pH in the range of about 6.7-7.2 suitablefor determining the identification of bacteria of the familyEnterobacteriaceae which comprises a chromogenic B-galactosidasesubstrate in combination with a member of the group consisting of (a) adecarboxylase substrate, (b) a deaminase substrate, (c) a ureasesubstrate, (d) a hydrogen sulfide detecting system, or (e) acarbohydrate fermentation system.

2. A culture medium of claim 1 wherein the chromogenic B-galactosidasesubstrate is chosen from the group consisting ofo-nitrophenyl-B-galactopyranoside,5-bromo-6-chloro-3-ind'olyl-,B-D-galactoside and 6-bromo-2-naphthyl-B-D-galactoside, the decarboxylase agents are lysine,ornithine and arginine, the urease substrate is urea and a carbohydratefermentation system selected from the group consisting of dextrose,mannitol, arabinose, sucrose, dulcitol, rhamnose, the deaminasesubstrate is selected from the group consisting or l-aminoacids,preferably tryptophane, phenylalanine, and histidine, and the hydrogensulfide detection system containing an iron salt in combination withhydrogen sulfide detecting agent preferably in the form of athiosulfate.

3. A culture medium of claim lwherein the chromogenic B-galactasidasesubstrate is in combination with a decarboxylase substrate.

4. A culture medium of claim 1 wherein the chromogenic B-galactosidasesubstrate is in combination with a deaminase substrate.

5. A culture medium of claim 1 wherein the chromo- 0 genieB-galactosidase substrate is in combination with a hydrogen sulfidedetecting system.

6. A culture medium of claim 1 wherein the chromo- 8. A culture mediumhaving a pH in the range of about 6.7-7.2 suitable for determining theidentification of bacteria of the Enterobacteriaceae which comprises thefollowing ingredients, said ingredients being present in proportionsindicated:

9. A process for preparing compositions of claim 8 which comprisesadmixing the bromthymol blue, yeast extract, dextrose, lysine, ferricammonium citrate, sodium thiosulfate, agar, lactose and sodium chlorideingredients in suitable quantities of water, adjusting the pH to about7.0, autoclaving the resulting solution followed by adding sterileo-nitrophenyl-ngalactopyranoside, urea and tryptophan, with q.s. waterto form the defined concentrations.

10. The process for the identification of bacteria of theEnterobacteriaceae which comprises inoculating the media defined byclaim 8 allowing the inoculated media to grow for at least about 24hours at 37C, followed by the identification of the bacteria accordingto the herein described changes for lysine, hydrogen sulfide,tryptophan, ONPG, urea and indole.

11. The process for the identification of bacteria of theEnterobacteriaceae which comprises inoculating the media defined byclaim 8 allowing the inoculated media to grow for at least about 24hours at 37C, followed b the idenification of the bacteria according tothe herein described color changed for glucose, lysine, hydrogensulfide, tryptophan, o-nitrophenyl ,B-galactopyranoside urea and indole.

12. A sterile culure medium having a pH of about Table-Continued Groupl: Hydrogen Sulfide Positive 6.7-7.2 comprising: H 5 Tryptophan lndoleLysine N G Urea l v P Salmonella lngredient Grams/liter Citrgbacfe v dKlebsl'ella or d Bromthymol Blue 0.05 E. cloacae d Yeast Extract 3.0 E.aeragenes Dextrose 0.9 E. hufniae or L-lysine 12.0

Ferric Ammonium Citrate 0.4 E. liquefaciens or d Sodium Thiosulfate 1.5Agar 12.0 Serratl'a d Lactose 0.02 Sodium Chloride 0.75 Differentbiochemical typesv O-nltrophenyl-B-galactopyranosrde 0.75 with exceptionof Slii ellu sonei. Tryptophan 3.75 or majority positive. Urea 1.0 -l--or majority negative. Water q.s. to make I000 cc.

16. The process of claim 14 wherein the color change r 13- A process forpreparing composltlon of clam 12, results are ead according to thefollowing chart which comprises admixing the bromthymo] blue, yeastextract, dextrose, lysine, ferric ammonium citrate, 50- COLOR REACTIONSOF ENTERUBACHiRMCEA/i WITH EXAMPLE rum thlosulfate, agar, lactose andsodium chloride ingredients in suitable quantities of water, adjustingthe I pH to about 7.0, autoclaving the resulting solution followed byadding sterile o-nitrophenyl-B- ORGANISMS SLANT BUTr gtgalactopyranoside urea and tryptophan, with q.s. water to form thedefined concentrations. (1) Providenc 'a Brown Yellow 14. The processfor the identification of bacteria of (2) g an Blue theEnterobacteriaceae which comprises inoculating 3 P, morganl'i do, Bluethe media defined by claim 12, allowing the inoculated (4) 'l'Blue/Black O (5) P. vulgarls do. Blue/Black media to grow for at leastabout 24 hours at 37 C, fol- (6) Klebsieua Green Blue/Green lowed by theidentification of the bacteria according to z qc r G Bl k/Y H the hereindescribed changes for lysine, hydrogen sul- (8) igi: gg f f tide,tryptophan, ONPG, urea and lndole. (9) Edwardsiella Blue Black/Green 15.The process of claim 14 wherein the color change (10 Green results areread according to the following table: (11) s l reen Green (12) E.liquefacl'ens Blue or Green or Table Green Yellow 4O (l3) E. aerogenesGreen Green (14) E. cloacae Green Yellow Group I: Hydrogen SullidcPositive (15) S. .t'lfllllll Green cllow H25 Tryptophan lndole LysineUrea (no as) (l6) Salmonella Blue Black (l7) Shi ella Blue Yellow orArizona m+ (18) E. lafniae Blue or Green Edwards-fella Green.S'ulnlmwllu I. ltll'rullilis Two colors in the butt. P. i'ulgarlsNumbers cross-correlate with FIGS. l and 2. Cilrubajler d reuli ii f I17. The process for the identification of bacteria of GroupTWPIOPhimPOsitlve (excludmg Group I Orgamsms) the Enterobacteriaceaewhich comprises inoculating H25 Tryptophan lndole Lysine Urea the mediadefined by claim 12, allowing the inoculated media to grow for at leastabout 24 hours at 37 C, followed b the identification of the bacteriaaccordin to P IIIOF'UIIH g the herein described color changes forglucose, lysine, Pftll'lllt'llllll hydrogen sulfide, tryptophan,o-nitrophenyl B-galactopyranoside urea and indole. Grou lll: lndolePositive (excludln Groups I and ll or anlsms) p g g 18. The processofclaim 17 wherein the color change "2 yp p lndflle Lysine Urea resultsare read according to the following chart:

E. (Oli shigellaz or COLOR REACTIONS OF ENTEROBAC I'ERIACEAE WITHKlehsialla or d EXAMPLE I Group IV: lndole Negative MEDIUM H 8Tryptophan lndole Lysine Urea Organisms Slant Butt lndole S/ligrlln or-F' J l Pm VllIl'IKll! Brown Yellow Continued I COLOR REACTIONS OFENTEROBACTERIACEAE WITH EXAMPLE 1 Organisms Slant Bun lndole (2) P.retlgeri Blue-brown to Olive Blue (3) P. morganii do. Blue (4) P.mirabilis do., Blue/Black (5) P. vulgaris do. Blue/Black (6) KlebsiellaGreen Blue/Green i (7) Cilrobncler freuna'ii Green Black/Yellow (8)Arizona Green Black/Green (9) Edwardsiella Blue Black/Green

1. A CULTURE MEDIUM HAVING A PH IN THE RANGE OF ABOUT 6.7-7.2 SUITABLEFOR DETERMINING THE IDENTIFICATION OF BACTERIA OF THE FAMILYENTEROBACTERIACEAE WHICH COMPRISES A CHROMOGENIC B-GALACTOSIDASESUBSTRATE IN COMBINATION WITH A MEMBER OF THE GROUP CONSISTING OF (A) ADECARBOXYLASE SUBSTRATE, (B) A DEAMINASE SUBSTRATE, (C) A UREASESUBSTRATE, (D) A HYDROGEN SULFIDE DETECTING SYSTEM, OR (E) ACARBOHYDRATE FERMENTATION SYSTEM.
 1. A culture medium having a pH in therange of about 6.7-7.2 suitable for determining the identification ofbacteria of the family Enterobacteriaceae which comprises a chromogenicBeta -galactosidase substrate in combination with a member of the groupconsisting of (a) a decarboxylase substrate, (b) a deaminase substrate,(c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e)a carbohydrate fermentation system.
 2. A culture medium of claim 1wherein the chromogenic Beta -galactosidase substrate is chosen from thegroup consisting of o-nitrophenyl- Beta -galactopyranoside,5-bromo-6-chloro-3-indolyl-Beta -D-galactoside and 6-bromo-2-naphthyl-Beta -D-galactoside, the decarboxylase agents are lysine, ornithine andarginine, the urease substrate is urea and a carbohydrate fermentationsystem selected from the group consisting of dextrose, mannitol,arabinose, sucrose, dulcitol, rhamnose, the deaminase substrate isselected from the group consisting or 1-aminoacids, preferablytryptophane, phenylalanine, and histidine, and the hydrogen sulfidedetection system containing an iron salt in combination with hydrogensulfide detecting agent preferably in the form of a thiosulfate.
 3. Aculture medium of claim 1 wherein the chromogenic Beta -galactasidasesubstrate is in combination with a decarboxylase substrate.
 4. A culturemedium of claim 1 wherein the chromogenic Beta -galactosidase substrateis in combination with a deaminase substrate.
 5. A culture medium ofclaim 1 wherein the chromogenic Beta -galactosidase substrate is incombination with a hydrogen sulfide detecting system.
 6. A culturemedium of claim 1 wherein the chromogenic Beta -galactosidase substrateis in combination with a urease substrate.
 7. A culture medium of claim1 wherein the chromogenic Beta -galactosidase substrate is incombination with a carbohydrate fermentation system.
 8. A culture mediumhaving a pH in the range of about 6.7-7.2 suitable for determining theidentification of bacteria of the Enterobacteriaceae which comprises thefollowing ingredients, said ingredients being present in proportionsindicated:
 9. A process for preparing compositions of claim 8 whichcomprises admixing the bromthymol blue, yeast extract, dextrose, lysine,ferric ammonium citrate, sodium thiosulfate, agar, lactose and sodiumchloride ingredients in suitable quantities of water, adjusting the pHto about 7.0, autoclaving the resulting solution followed by addingsterile o-nitrophenyl- Beta -galactopyranoside, urea and tryptophan,with q.s. water to form the defined concentrations.
 10. The process forthe identification of bacteria of the Enterobacteriaceae which comprisesinoculating the media defined by claim 8 allowing the inoculated mediato grow for at least about 24 hours at 37*C, followed by theidentification of the bacteria according to the herein described changesfor lysine, hydrogen sulfide, tryptophan, ONPG, urea and indole.
 11. Theprocess for the identification of bacteria of the Enterobacteriaceaewhich comprises inoculating the media defined by claim 8 allowing theinoculated media to grow for at least about 24 hours at 37*C, followed bthe idenification of the bacteria according to the herein describedcolor changed for glucose, lysine, hydrogen sulfide, tryptophan,o-nitrophenyl Beta -galactopyranoside urea and indole.
 12. A sterileculure medium having a pH of about 6.7-7.2 comprising:
 13. A process forpreparing composition of claim 12, which comprises admixing thebromthymol blue, yeast extract, dextrose, lysine, ferric ammoniumcitrate, sodium thiosulfate, agar, lactose and sodium chlorideingredients in suitable quantities of water, adjusting the pH to about7.0, autoclaving the resulting solution followed by adding sterileo-nitrophenyl- Beta -galactopyranoside urea and tryptophan, with q.s.water to form the defined concentrations.
 14. The process for theidentification of bacteria of the Enterobacteriaceae which comprisesinoculating the media defined by claim 12, allowing the inoculated mediato grow for at least about 24 hours at 37*C, followed by theidentification of the bacteria according to the herein described changesfor lysine, hydrogen sulfide, tryptophan, ONPG, urea and indole.
 15. Theprocess of claim 14 whErein the color change results are read accordingto the following table:
 16. The process of claim 14 wherein the colorchange results are read according to the following chart:
 17. Theprocess for the identification of bacteria of the Enterobacteriaceaewhich comprises inoculating the media defined by claim 12, allowing theinoculated media to grow for at least about 24 hours at 37*C, followedby the identification of the bacteria according to the herein describedcolor changes for glucose, lysine, hydrogen sulfide, tryptophan,o-nitrophenyl Beta -galactopyranoside urea and indole.